Hop (scientific name: Humulus lupulus) is a dioecious climbing plant belonging to the Cannabaceae family. The female plant of hop produces flower-like cones, called strobilus, like those of a pine tree, and these cones are used as an ingredient of beer. Unfertilized cones contain yellow grains called lupulin, which give the unique flavor and refreshing bitterness of beer. Hop is one of the essential ingredients for brewing beer in that it has various functions, such as imparting bittersweet and refreshing flavor, improving froth retention, improving the luster of beer, and reducing the growth of saprophytic bacteria.
Hops are cultivated mainly in various countries, including Germany, the U.S., and other countries such as Czech, the U.K., France, China, Slovenia, South Africa, Australia, New Zealand, and Japan, and the varieties of hops cultivated are wide-ranging. The varieties of hops are broadly divided into two categories: aromatic hops and bittering hops. Aromatic hops impart luxurious flavor and mild bitterness, and bittering hops give crisp bitterness. As such, hops produce different flavors and tastes depending on the variety, so in order to brew high-quality beer, it is important to select a hop variety suited to the purpose. Further, in order to maintain the quality of beer in a stable manner, it is important to strictly check to see if hops as a raw material are delivered correctly as ordered. Hitherto, checking has been made on the basis of quality assurance certificates provided by suppliers, the differences in the contents of various components including α-acid in lupulin, or the difference in the appearance of cones. However, the hop cones commonly purchased and used for brewing beers in Japan are those which have been dried, crushed, and compressed into pellets in advance. Thus, it is very difficult to identify hop varieties from the appearance characteristics of their cones. In addition, chemical components obtained from cones, such as bittering components and essential oil components, are susceptible to environmental factors, and those components from some varieties show similar values; so there has been a limit to using those components as indicators for identification.
Meanwhile, there have in recent years been developed several variety identification methods to which genetic engineering is applied. These methods identify varieties by detecting a difference in nucleotide sequence among the varieties, in other words polymorphism. In the field of hops, International Patent Publication No. WO 1997/005281 discloses a hop variety identification method in which a DNA region that differs among varieties is detected by the RAPD method, primers capable of amplifying the region are designed, the polymorphic region is amplified by PCR using the primers, and the presence or absence of amplified fragments, or the difference in size among them, is analyzed, whereby the hop varieties are identified. Japanese Patent Application Publication No. JP H11-103895 also discloses a hop variety discrimination method in which a genomic DNA suitable for PCR is efficiently extracted and purified from a hop by using a high concentration of lithium chloride, the genomic DNA is subjected to PCR using primers such as random primers and STS primers, to thereby amplify a DNA that enables identification of the variety of the hop, and the hop variety is discriminated according to the presence or absence of an amplified DNA. Japanese Patent Application Publication No. JP 2006-34142 further discloses a variety identification method in which a microsatellite DNA region containing a polymorphism among varieties is amplified by performing PCR using primers designed to be capable of amplifying the region, and a difference in length among amplified DNA fragments is analyzed to identify the varieties.
The above-mentioned methods, in which hop varieties are identified using a polymorphism(s) on hop genomes, are effective in that they allow exact identification of hop varieties without being affected by the cultivation environment, harvest timing, or storage method of hops. However, since these methods all make identification based on the presence or absence of amplification by PCR, or on a difference in amplification product size, these are susceptible to variations in conditions for enzymatic reaction, electrophoresis, gel staining, or the like, and are problematic in reproducibility. In other words, it is difficult to distinguish whether no band appears due to any failure of PCR or staining, or no amplification occurs originally, so there is a risk of misinterpreting the results. Further, the above-mentioned methods detect DNA fragments by capillary electrophoresis and laser irradiation, by staining of samples for gel electrophoresis, or by other similar means; thus, though they can at least identify the varieties of hops to be tested or deduce the presence or absence of mixing of a different variety of hop, they cannot go so far as to deduce or identify the variety of the hop mixed or to analyze the proportion of the hop variety mixed. Furthermore, in a case where a DNA is fragmented due to, for example, deterioration of or damage to a test substance, there occurs a decrease in accuracy of analysis, thereby posing a risk of misinterpreting the results.
Single nucleotide polymorphism (hereinafter also referred to as “SNP”) is attracting attention as a very useful and easy-to-use polymorphic marker. SNP is a polymorphism in nucleotide sequence caused by a difference at a single nucleotide. SNP is insusceptible to variations in reaction conditions, and hence produces no false results and is easy to determine. Further, SNP data can be converted to a digital signal consisting of 0 and 1 and be information-processed, so that sample processing and data analysis can be automated easily and also various analyses can be made using an enormous amount of SNP data. Furthermore, it is possible to make many SNP analyses at one time, for example, through designing primers with non-specific tails of different lengths.
However, hop has 20 diploid chromosomes (10 pairs of double strands) and a huge genome size of about 2.7 to 3 billion base pairs (2.7-3 GB), and also has not been made clear as to its relationship with its structural genes. Therefore, from the results of genomic analysis, there has not been found such a sufficient number of SNPs among hop varieties that allow identification of many varieties, and there has not yet been known a method for identifying hop varieties using a SNP as a polymorphic marker.